1. Field of the Invention
This disclosure generally involves manufacturing plasma-derived therapeutic protein solutions, and more specifically, manufacturing selectively stabilized animal or human serum albumin (HSA), Alpha-1 Protease Inhibitor (Alpha-1 PI), and Antithrombin III (AT III) from serum or plasma.
2. Prior Art Description
The Cohn fractionation method, which utilizes ethanol, temperature, pH, protein concentration, ionic strength, and time to insolubilize unwanted proteins during albumin manufacture, was originally published in 1946, and remains a primary method in the United States for processing plasma. Cohn et al., J. Am. Chem. Soc. 68, 459 (1946). T. Gerelough's subsequent use of 95% ethanol in the Cohn fractionation process greatly diminished process volumes required, and thereby reduced corresponding manufacturing costs. Gerelough's method is also a recognized standard in the United States for plasma fractionation. U.S. Pat. Nos. 2,710,294; 2,710,293 (1955). In Europe, H. Nitschamann and P. Kistler describe a shorter method for processing albumin; however, the resulting product failed to satisfy regulatory guidelines imposed by United States agencies during that time period. Vox Sang., 5, 272 (1960).
Plasma fractionation methods in the United States have employed Cohn techniques since Cohn et al's. 1946 publication. Consequently, neither interest nor necessity encouraged manufacturers to identify alternative albumin fractionation techniques for more than twenty years. M. Steinbuch, Vox Sang., 23, 92 (1972). Furthermore, since conventional fractionation techniques produced albumin that could be successfully pasteurized (by heating for 10 hours at 60 degrees centigrade) to inactivate viruses, there was little motivation and much caution among albumin manufacturers in seeking alternative and improved fractionation methods.
Then in 1972 M. Steinbuch explored the ability of several reagents, other than ethanol, to separate plasma proteins via precipitation. Using Cohn Fraction III as starting material, Steinbuch studied the precipitation capacity of caprylic acid, which had previously been used to stabilize albumin (M. Steinbuch, Vox Sang. 23:92-106, 1972, Yu L. Hao, U.S. Pat. No. 4,222,934, 1980), and subsequently to inactivate lipid-enveloped viruses. Seng et al., U.S. Pat. No. 4,939,176, 1990. As a result of these studies, scientists later developed several techniques for purifying IgG, IgA, alpha-1 acid glycoprotein and prealbumin, concurrently finding that the precipitation reaction was highly temperature, and pH dependent.
During human immunoglobulin preparation caprylic acid is generally recognized as an effective precipitating agent for most plasma proteins at pH 4.8, so long as parameters such as temperature and ionic strength are optimized. Steinbuch et al., Preparative Biochemistry, 3(4), 363-373 (1973). Accordingly, Steinbuch et al. have described a method for isolating IgG from mammalian sera using caprylic acid, finding that extensive non-immunoglobulin precipitation is best obtained at slightly acidic ph, but not below pH 4.5. Steinbuch et al., Arch. Biochem. Biophys., 134, 279-294 (1969).
Habeeb et al. used caprylic acid precipitation to obtain plasma-derived IgG that was free of aggregates, plasmin and plasminogen; low in anticompliment activity; and stable during storage. Preparative Biochemistry, 14(1), 1-17 (1984). Also, IgA has been prepared as a routine fractionation by-product from Cohn fraction III, based on IgA solubility with caprylic acid present at pH 4.8. Pejaudier et al., Vox Sang. 23, 165-175 (1972). Fraction III additionally provides starting material for obtaining IgM-enriched plasma fractions.
Sodium caprylate has also been used to purify albumin. According to these methods, sodium caprylate is added to process plasma, and protects albumin when the process stream is exposed to high temperatures. Extreme temperatures not only denature process stream globulins, but often generate contaminant neo-antigens. Schneider et al., U.S. Pat. No. 4,156,681 (1979); Institute Merieux, U.S. Pat. No. 3,992,367.
Cohn fraction III was also treated with caprylic acid to precipitate ceruloplasmin, an alpha globulin that facilitates plasma copper transport. Employing this technique to obtain ceruloplasmin avoided denaturation steps involving ethanol or acetone, but has been obtained in this way only from horse, mule, rabbit, goat, sheep, and baboon plasma. M. Steinbuch, Vox Sang., 23, 92-106 (1972).
Currently, technical and patent literature contain numerous albumin manufacturing methods that incorporate sodium caprylate as a stabilizing agent, and purification techniques involving caprylic acid precipitation to obtain immunoglobulins from plasma-derived Cohn fraction III. However, we are unaware of disclosures employing sodium caprylate as a partitioning agent during albumin manufacture to separate albumin from unwanted globulins and manufacturing debris.
Unexpectedly, we have also discovered significant advantages in manufacturing albumin with sodium caprylate to partition albumin from unwanted material, instead of using an ethanol precipitant. First, sodium caprylate partitioning shortens conventional albumin manufacturing methods, which reduces product handling and correspondingly improves albumin recovery by 25% or more. Resulting albumin yields are essentially aluminum-free, and exhibit 97% or greater monomer levels. Second, sodium caprylate partitioning significantly reduces the time needed to complete albumin manufacture, thus reducing equipment-related manufacturing costs by at least 65%. Third, sodium caprylate partitioning improves Alpha-1 PI and AT-III yields from Cohn fraction II+III, and is generally more energy efficient than conventional methods of manufacturing plasma products. Finally, and perhaps most important, sodium caprylate partitioning significantly reduces ethanol use, and completely eliminates the use of acetone during albumin manufacture, which largely avoids polluting our environment with noxious and environmentally harmful solvent residues. Our findings are discussed and illustrated in detail below.